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Ex situ microelectrode experiments, using cyanobacterial biofilms from karst water creeks, were conducted under various pH, temperature, and constant-alkalinity conditions to investigate the effects of changing environmental parameters on cyanobacterial photosynthesis-induced calcification. Microenvironmental chemical conditions around calcifying sites were controlled by metabolic activity over a wide range of photosynthesis and respiration rates, with little influence from overlying water conditions. Regardless of overlying water pH levels (from 7.8 to 8.9), pH at the biofilm surface was approximately 9.4 in the light and 7.8 in the dark. The same trend was observed at various temperatures (4°C and 17°C). Biological processes control the calcium carbonate saturation state (Ω) in these and similar systems and are able to maintain Ω at approximately constant levels over relatively wide environmental fluctuations. Temperature did, however, have an effect on calcification rate. Calcium flux in this system is limited by its diffusion coefficient, resulting in a higher calcium flux (calcification and dissolution) at higher temperatures, despite the constant, biologically mediated pH. The ability of biological systems to mitigate the effects of environmental perturbation is an important factor that must be considered when attempting to predict the effects of increased atmospheric partial CO 2 pressure on processes such as calcification and in interpreting microfossils in the fossil record.

Sulfidic seeps with methane ebullition were observed at the low-water line of intertidal sand flats at a number of locations in the Wadden Sea. Bioturbating fauna was absent in the seep areas but abundant in the more central areas of the tidal flat. At one site, the vertical methane and sulfate distribution in pore water was determined along transects from the low-water line toward the interior of the sand flat. The resulting two-dimensional distributions showed a plume of methane-rich and sulfate-depleted pore water reaching from a depth below 1.2 m beneath the sand surface up to the sediment surface at the low-water line. The δ13C of methane released at the seeps was -68.6‰, indicating a biological origin. The 14C signature of methane was clearly elevated by anthropogenic radiocarbon, which shows that the methane was formed less than 50 yr ago. The observations indicate an internal circulation, where water enters the sand flats in the central area and exits at the low-water line. Pore-water flow patterns in the sand flat during the tidal cycle were calculated from the surface topography and from the pressure distribution at the flat surface across the tidal cycle. The calculated flow patterns explain the measured methane and sulfate distributions and predict a residence time of the seepage water of about 30 yr. Intertidal sand flats act as one-way valves, passing water from the central surface through the interior of the flat to an outflow zone at and below the low-water line with a velocity of millimeters to centimeters per day. The flow causes permeable tidal flats to emit methane to the surface water and atmosphere in substantial amounts. © 2008, by the American Society of Limnology and Oceanography, Inc.

Pigment analysis in an intact hypersaline microbial mat by hyperspectral imaging revealed very patchy and spatially uncorrelated distributions of photopigments Chl a and BChl a/c, which are characteristic photopigments for oxygenic (diatoms and cyanobacteria) and anoxygenic phototrophs (Chloroflexaceae). This finding is in contrast to the expectation that these biomarker pigments should be spatially correlated, as oxygenic phototrophs are thought to supply the Chloroflexaceae members with organic substrates for growth. We suggest that the heterogeneous occurrence is possibly due to sulfide, whose production by sulfate-reducing bacteria may be spatially heterogeneous in the partially oxic photic zone of the mat. We furthermore mapped the near-infra-red-light controlled respiration of Chloroflexaceae under light and dark conditions and found that Chloroflexaceae are responsible for a major part of oxygen consumption at the lower part of the oxic zone in the mat. The presence of Chloroflexaceae was further confirmed by FISH probe and 16S rRNA gene clone library analysis. We assume that species related to the genera Oscillochloris and 'Candidatus Chlorothrix', in contrast to those related to Chloroflexus and Roseiflexus, depend less on excreted photosynthates but more on the presence of free sulfide, which may explain their presence in deeper parts of the mat.

Microsensors are powerful tools for microenvironment studies, however their use has often been restricted to laboratory applications due to the lack of adequate equipment for in situ deployments. Here we report on new features, construction details, and examples of applications of an improved diver-operated motorized microsensor profiler for underwater field operation to a water depth of 25 m. The new motorized profiler has a final precision of 5 microm, and can accommodate amperometric Clark-type microsensors for oxygen and hydrogen sulfide, potentiometric microsensors (e.g., for pH, Ca2+), and fiber-optic irradiance microsensors. The profiler is interfaced by a logger with a signal display, and has pushbuttons for underwater operation. The system can be pre-programmed to autonomous operation or interactively operated by divers. Internal batteries supply power for up to 24 h of measurements and 36 h of data storage (max. 64 million data points). Two flexible stands were developed for deployment on uneven or fragile surfaces, such as coral reefs. Three experimental pilot studies are presented, where (1) the oxygen distribution in a sand ripple was 3-D-mapped, (2) the microenvironment of sediment accumulated on a stony coral was studied, and (3) oxygen dynamics during an experimental sedimentation were investigated. This system allows SCUBA divers to perform a wide array of in situ measurements, with deployment precision and duration similar to those possible in the laboratory.

The aim of this study was to investigate the supposed vertical diel migration and the accompanying physiology of Beggiatoa bacteria from hypersaline microbial mats. We combined microsensor, stable-isotope, and molecular techniques to clarify the phylogeny and physiology of the most dominant species inhabiting mats of the natural hypersaline Lake Chiprana, Spain. The most dominant morphotype had a filament diameter of 6 to 8 μm and a length varying from 1 to >10 mm. Phylogenetic analysis by 16S rRNA gene comparison revealed that this type appeared to be most closely related (91% sequence identity) to the narrow (4-μm diameter) nonvacuolated marine strain MS-81-6. Stable-isotope analysis showed that the Lake Chiprana species could store nitrate intracellularly to 40 mM. The presence of large intracellular vacuoles was confirmed by fluorescein isothiocyanate staining and subsequent confocal microscopy. In illuminated mats, their highest abundance was found at a depth of 8 mm, where oxygen and sulfide co-occurred. However, in the dark, the highest Beggiatoa densities occurred at 7 mm, and the whole population was present in the anoxic zone of the mat. Our findings suggest that hypersaline Beggiatoa bacteria oxidize sulfide with oxygen under light conditions and with internally stored nitrate under dark conditions. It was concluded that nitrate storage by Beggiatoa is an optimal strategy to both occupy the suboxic zones in sulfidic sediments and survive the dark periods in phototrophic mats.

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments., Author Summary In 1888 Winogradsky proposed the concept of chemolithotrophy—growth using inorganic compounds as an energy source—after studying the sulfur bacterium Beggiatoa. These filamentous bacteria and related organisms inhabit the surface of marine and freshwater sediments, where they oxidize hydrogen sulfide using either oxygen or nitrate. In particular, conspicuously large marine representatives accumulate nitrate in vacuoles to survive anoxia, a unique feature among prokaryotes. Since nitrate-storing Beggiatoa are not available in pure culture, we amplified and sequenced the genomic DNA of single multicellular filaments. We comprehensively tested the incomplete sequence assemblies for foreign DNA. We show that the Beggiatoa genome encodes the pathways of chemolithoautotrophy but also appears to support the use of alternative electron donors and acceptors. We propose that vacuolar-type ATPases generate an electrochemical gradient to drive nitrate transport over the vacuole membrane, a mechanism similar to eukaryotic solute accumulation. Intriguingly, we found evidence for substantial gene exchange between Beggiatoa and cyanobacteria. In both phyla, hemagglutinins are possibly involved in filament formation. The breadth of storage and metabolic capabilities encoded in its genome enables Beggiatoa to act as a “rechargeable battery,” which glides between oxic and sulfidic zones to overcome non-overlapping availabilities of electron donors and acceptors., Examining the genome of single filaments of Beggiatoa deepens our understanding of their contribution to sulfur and nitrogen cycling in marine sediments.

The effects of salinity fluctuation on bacterial diversity, rates of gross photosynthesis (GP) and oxygen consumption in the light (OCL) and in the dark (OCD) were investigated in three submerged cyanobacterial mats from a transect on an intertidal flat. The transect ran 1 km inland from the low water mark along an increasingly extreme habitat with respect to salinity. The response of GP, OCL and OCD in each sample to various salinities (65 per thousand, 100 per thousand, 150 per thousand and 200 per thousand) were compared. The obtained sequences and the number of unique operational taxonomic units showed clear differences in the mats' bacterial composition. While cyanobacteria decreased from the lower to the upper tidal mat, other bacterial groups such as Chloroflexus and Cytophaga/Flavobacteria/Bacteriodetes showed an opposite pattern with the highest dominance in the middle and upper tidal mats respectively. Gross photosynthesis and OCL at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP, OCL and OCD rates at salinities > 100 per thousand. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.

Mud volcanism is an important natural source of the greenhouse gas methane to the hydrosphere and atmosphere(1,2). Recent investigations show that the number of active submarine mud volcanoes might be much higher than anticipated ( for example, see refs 3 - 5), and that gas emitted from deep-sea seeps might reach the upper mixed ocean(6-8). Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release are unknown(9). It is also unclear how efficiently methane-oxidizing microorganisms remove methane. Here we investigate the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72 degrees N, 14 degrees 44' E; 1,250 m water depth) to provide quantitative estimates of the in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities: aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a previously undescribed clade of archaea (ANME-3) associated with bacterial mats. We found that the upward flow of sulphate- and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to

The diversity and function of aerobic heterotrophic bacteria (AHB) in cyanobacterial mats have been largely overlooked. We used culture-dependent and molecular techniques to explore the species diversity, degradative capacities and functional guilds of AHB in the photic layer (2 mm) of an oil-polluted microbial mat from Saudi Arabia. Enrichment isolation was carried out at different salinities (5% and 12%) and temperatures (28 and 45 °C) and on various substrates (acetate, glycolate, Spirulina extract and crude oils). Counts of most probable number showed a numerical abundance of AHB in the range of 1.15–8.13×106 cells g−1 and suggested the presence of halotolerant and thermotolerant populations. Most of the 16S rRNA sequences of the obtained clones and isolates were phylogenetically affiliated to the groups Gammaproteobacteria, Bacteriodetes and Alphaproteobacteria. Groups like Deltaproteobacteria, Verrucomicrobia, Planctomycetes, Spirochaetes, Acidobacteria and Deinococcus-Thermus were only detected by cloning. The strains isolated on acetate and glycolate belonged to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains enriched on crude oil belonged to Marinobacter and Alcanivorax. Members of the Bacteriodetes group were only enriched on Spirulina extract indicating their specialization in the degradation of cyanobacterial dead cells. The substrate spectra of representative strains showed the ability of all AHB to metabolize cyanobacterial photosynthetic and fermentation products. However, the unique in situ conditions of the mat apparently favored the enrichment of versatile strains that grew on both the cyanobacterial exudates and the hydrocarbons. We conclude that AHB in cyanobacterial mats represent a diverse community that plays an important role in carbon-cycling within microbial mats.

Microbial mats are characterized by high primary production but low growth rates, pointing to a limitation of growth by the lack of nutrients or substrates. We identified compounds that instantaneously stimulated photosynthesis rates and oxygen consumption rates in a hypersaline microbial mat by following the short-term response (c. 6 h) of these processes to addition of nutrients, organic and inorganic carbon compounds, using microsensors. Net photosynthesis rates were not stimulated by compound additions. However, both gross photosynthesis and oxygen consumption were substantially stimulated (by a minimum of 25%) by alanine (1 mM) and glutamate (3.5 mM) as well as by phosphate (0.1 mM). A low concentration of ammonium (0.1 mM) did not affect photosynthesis and oxygen consumption, whereas a higher concentration (3.5 mM) decreased both process rates. High concentrations of glycolate (5 mM) and phosphate (1 mM) inhibited gross photosynthesis but not oxygen consumption, leading to a decrease of net photosynthesis. Photosynthesis was not stimulated by addition of inorganic carbon, nor was oxygen consumption stimulated by organic compounds like glycolate (5 mM) or glucose (5 mM), indicating that carbon was efficiently cycled within the mat. Photosynthesis and oxygen consumption were apparently tightly coupled, because stimulations always affected both processes to the same extent, which resulted in unchanged net photosynthesis rates. These findings illustrate that microsensor techniques, due to their ability to quantify all three processes, can clarify community responses to nutrient enrichment studies much better than techniques that solely monitor net fluxes.

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH 3 and HNO 2 ) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low K m s for ammonia ( Nitrosopira spp., Nitrosomonas oligotropha , and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.

IMPACT OF SULFIDE ON NITRATE CONVERSION IN EUTROPHIC NITRATE-RICH MARINE SLUDGEC.U. Schwermer 1, B.U. Krieger 2, G. Lavik 1, A. Schramm 3, J. van Rijn 4, D. de Beer 1, D. Minz 5, E. Cytryn 4, M. Kuypers 1, A. Gieseke 11 Max Planck Institute for Marine Microbiology, Bremen, Germany; 2 Dept. of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany; 3 Dept. of Biological Sciences, Microbiology, University of Aarhus, Denmark; 4 Faculty of Agricultural, Food And Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel; 5 Institute for Soil, Water and Environmental Sciences, Volcani Research Center, Bet-Dagan, IsraelMultiple anaerobic processes are responsible for carbon mineralization in eutrophic nitrate-rich marine environments (e.g., upwelling areas, estuaries, and aquacultures), involving electron acceptors from both the nitrogen and sulfur cycle. The interaction of these processes is less understood. Our aim was to investigate the functional interaction of nitrate reduction, denitrification and sulfate reduction in an anaerobic marine sludge. We hypothesize that sulfide (from sulfate reduction) (i) causes incomplete denitrification, and (ii) directs nitrate conversion from denitrification to dissimilatory nitrate-reduction to ammonium (DNRA). In situ microsensor profiling in stagnant sludge revealed the typical stratification of nitrate reduction on top of sulfate reduction. Increasing the bulk nitrate concentration lead to a downward shift of the nitrate-sulfide transition zone; within this zone, N2O accumulated to 260 µM. Batch incubations of sludge with sulfide and 15N-nitrate showed the formation of ammonium at the expense of N2 with increasing sulfide concentrations; an indication of the predicted shift from denitrification to DNRA. However, at the highest sulfide concentration tested (1.6 mM), only 22% and 20% of the nitrate added were converted to N2 and ammonium, respectively, while N2O became the main end product (56%). These findings were corroborated by pure culture experiments with nitrate-reducing and denitrifying isolates from the sludge. Our results show that the presence of sulfide generally decreased growth rates but increased N2O production. We conclude that sulfide plays a key role in causing incomplete denitrification, presumably by inhibiting the N2O reductase, and enhancing DNRA compared to denitrification.

Eight anaerobic granular sludges were surveyed for Crenarchaeota using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3 Crenarchaeota represented up to 50% of Archaea and 25% of the total microbiota in five sludges. Crenarchaeota were localized in close association with methanogenic Archaea.

The spatial heterogeneity of photosynthesis and calcification of single polyps of the coral Galaxea fascicularis was investigated. Photosynthesis was investigated with oxygen microsensors. The highest rates of gross photosynthesis (Pg) were found on the tissue covering the septa, the tentacles, and the tissues surrounding the mouth opening of the polyp. Lower rates were found on the tissues of the wall and the coenosarc. Calcification was investigated by radioactive tracers. The incorporation pattern of 45Ca and 14C in the corallites was imaged with use of a Micro-Imager. The β-images obtained showed that the incorporation of the radioactive tracers coincided with the Pg distribution pattern with the highest incorporation rates found in the corallite septa. Thus, the high growth rate of the septa is supported by the high rates of Pg by the symbiont in the adjacent tissues. The total incorporation rates were higher in light than in dark, however, the distribution pattern of the radioisotope incorporation was not affected by illumination. This further emphasizes the close relation between calcification and photosynthesis.

Two freshwater sediments (organic-poor and organic-rich) that contained their distinct natural microbial communities were incubated in experimental microcosms with either NH4 + or NO2 - in the overlying water. Microsensor measurements revealed the thin oxic surface layer as a site of initially high rates of nitrification, i.e. O2, NH4 +, and NO2 - consumption, and NO3 - production. Unexpectedly, during the subsequent 4-week incubation NH4 + consumption decreased in both sediment types and NO2 - consumption decreased in the organic-rich sediment. In the organic-rich sediment O2 consumption and NO3 - production paralleled these decreases, i.e. the reduced NH4 + and NO2 - consumption rates were most probably due to reduced activity of nitrifiers. These microsensor data imply factors other than frequently suggested competition between nitrifiers and heterotrophs for NH4 +, NO 2 - or O2 as causes for the loss of nitrification activity. We hypothesize that experimental manipulations (e.g. removal of macrofauna, redistribution of particulate organic matter, permanent nutrient enrichment) rendered the performance of the microbial community unstable. It is thus recommendable to restrict experiments in such commonly used model systems to the period of highest stability.

The benthic microbial mat community of the only permanent hypersaline natural inland lake of Western Europe, ‘La Salada de Chiprana’, northeastern Spain, was structurally and functionally analyzed. The ionic composition of the lake water is characterized by high concentrations of magnesium and sulfate, which were respectively 0.35 and 0.5 M at the time of sampling while the total salinity was 78 g l-1. Community composition was analyzed by microscopy, high-performance liquid chromatography (HPLC) pigment analyses and by studying culturable bacteria from different functional groups. Therefore, denaturing gradient gel electrophoresis (DGGE) was applied on most probable number (MPN) dilution cultures. Microscopy revealed that a thin layer of Chloroflexus-like bacteria overlaid various cyanobacteria-dominated layers each characterized by different morphotypes. DGGE analysis of MPN dilution cultures from distinct mat layers showed that various phylotypes of anoxygenic phototrophic, aerobic heterotrophic, colorless sulfur-, and sulfate-reducing bacteria were present. The mats were furthermore functionally studied and attention was focussed on the relationship between oxygenic primary production and the flow of carbon through the microbial community. Microsensor techniques, porewater and sediment photopigment analysis were applied in order to estimate oxygenic photosynthetic rates, daily dynamics of (in)organic carbon porewater concentration and migration behavior of phototrophs. Chiprana microbial mats produced dissolved organic carbon (DOC) both during the day and night. It was estimated that 14% of the mats gross photosynthetic production and 49% of the mats net photosynthetic production diffused out of the mat in the form of low molecular mass fatty acids, although these compounds made up only 2% of the total DOC pool. The high flux of dissolved fatty acids from the microbial mat to the water column may explain why in this system Chloroflexus-like bacteria proliferate on top of the cyanobacterial layers since these photoheterotrophic bacteria grow preferably on organic phototrophic exudates. Furthermore it may also explain why high numbers of viable sulfate-reducing bacteria were found in the fully oxygenated sediment surface layers. These organisms apparently do not have to compete with aerobic heterotrophic community members due to the ample availability of organic substrates. Moreover, the high production of DOC strongly indicates that the mat community was nutrient limited in its growth. Photopigment analysis revealed furthermore that chlorophyll a (Chla) and three of its allomeres had a complementary depth distribution what suggests that the Chla allomeres are functional adaptations to differences in light quality and/or quantity and may be species specific.

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mumol photons/m(2)s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.

In this study the degradation of hydrophobic petroleum model compounds (phenanthrene, pristane, octadecane and dibenzothiophene) added to a submersed hypersaline microbial mat was investigated. Montmorillonite with an artificially altered, hydrophobic surface was used as carrier material, forming an organo-clay complex (OCC) with the attached mixture of petroleum model compounds. 6 mg/cm2 OCC were applied to cyanobacterial mat pieces, containing approximately 33.3 microg/mg OCC of each compound. The degradation experiment was performed under controlled laboratory conditions and accompanied by chemical analyses by GC/GC-MS, molecular analyses by PCR and DGGE as well as functional analyses by microsensor measurements of oxygen, photosynthesis, sulfide, pH and light. All applied model compounds were degraded, but residues were still present after 18 weeks. The aromatic compounds phenanthrene (5.1 microg/mg OCC) and dibenzothiophene (4.3 microg/mg OCC) were preferentially degraded compared to the alkanes pristane (12.4 microg/mg OCC) and n-octadecane (13.4 microg/mg OCC). Metabolic changes during the degradation process could not be detected by microsensor measurements. The molecular population analyses did not reveal any significant community changes concomitant with the decrease of the petroleum model compounds. We conclude, that the pristine mats represent an intact, robust ecosystem in which the enzymatic requirements for the degradation of the applied pollutants exist. The slow degradation process did not affect the usual high internal turnover rates and did not favor a certain population in the community of the mats.

A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.

We examined soil surface colour change to green and hydrotaxis following addition of water to biological soil crusts using pigment extraction, hyperspectral imaging, microsensors and C-13 labeling experiments coupled to matrix-assisted laser desorption and ionization time of flight-mass spectrometry (MALD-TOF MS). The topsoil colour turned green in less than 5 minutes following water addition. The concentrations of chlorophyll a (Chl a), scytonemin and echinenon rapidly increased in the top,1 mm layer while in the deeper layer, their concentrations remained low. Hyperspectral imaging showed that, in both wet and dehydrated crusts, cyanobacteria formed a layer at a depth of 0.2-0.4 mm and this layer did not move upward after wetting. C-13 labeling experiments and MALDI TOF analysis showed that Chl a was already present in the desiccated crusts and de novo synthesis of this molecule started only after 2 days of wetting due to growth of cyanobacteria. Microsensor measurements showed that photosynthetic activity increased concomitantly with the increase of Chl a, and reached a maximum net rate of 92 mu mol m(-2) h(-1) approximately 2 hours after wetting. We conclude that the colour change of soil crusts to green upon water addition was not due to hydrotaxis but rather to the quick recovery and reassembly of pigments. Cyanobacteria in crusts can maintain their photosynthetic apparatus intact even under prolonged periods of desiccation with the ability to resume their photosynthetic activities within minutes after wetting.

The Um Alhool area in Qatar is a dynamic evaporative ecosystem that receives seawater from below as it is surrounded by sand dunes. We investigated the chemical composition, the microbial activity and biodiversity of the four main layers (L1-L4) in the photosynthetic mats. Chlorophyll a (Chl a) concentration and distribution (measured by HPLC and hyperspectral imaging, respectively), the phycocyanin distribution (scanned with hyperspectral imaging), oxygenic photosynthesis (determined by microsensor), and the abundance of photosynthetic microorganisms (from 16S and 18S rRNA sequencing) decreased with depth in the euphotic layer (L1). Incident irradiance exponentially attenuated in the same zone reaching 1% at 1.7-mm depth. Proteobacteria dominated all layers of the mat (24%-42% of the identified bacteria). Anoxygenic photosynthetic bacteria (dominated by Chloroflexus) were most abundant in the third red layer of the mat (L3), evidenced by the spectral signature of Bacteriochlorophyll as well as by sequencing. The deep, black layer (L4) was dominated by sulfate reducing bacteria belonging to the Deltaproteobacteria, which were responsible for high sulfate reduction rates (measured using 35S tracer). Members of Halobacteria were the dominant Archaea in all layers of the mat (92%-97%), whereas Nematodes were the main Eukaryotes (up to 87%). Primary productivity rates of Um Alhool mat were similar to those of other hypersaline microbial mats. However, sulfate reduction rates were relatively low, indicating that oxygenic respiration contributes more to organic material degradation than sulfate reduction, because of bioturbation. Although Um Alhool hypersaline mat is a nutrient-limited ecosystem, it is interestingly dynamic and phylogenetically highly diverse. All its components work in a highly efficient and synchronized way to compensate for the lack of nutrient supply provided during regular inundation periods. Qatar University, fund number QUUG-CAS-BES-11/12-, and Max-Planck Institute for Marine Microbiology. Scopus

Background: A wealth of microbial eukaryotes is adapted to life in oxygen-deficient marine environments. Evidence is accumulating that some of these eukaryotes survive anoxia by employing dissimilatory nitrate reduction, a strategy that otherwise is widespread in prokaryotes. Here, we report on the anaerobic nitrate metabolism of the fungus Aspergillus terreus (isolate An-4) that was obtained from sediment in the seasonal oxygen minimum zone in the Arabian Sea, a globally important site of oceanic nitrogen loss and nitrous oxide emission. Results: Axenic incubations of An-4 in the presence and absence of oxygen and nitrate revealed that this fungal isolate is capable of dissimilatory nitrate reduction to ammonium under anoxic conditions. A 15N-labeling experiment proved that An-4 produced and excreted ammonium through nitrate reduction at a rate of up to 175 nmol 15NH4 + g-1 protein h-1. The products of dissimilatory nitrate reduction were ammonium (83%), nitrous oxide (15.5%), and nitrite (1.5%), while dinitrogen production was not observed. The process led to substantial cellular ATP production and biomass growth and also occurred when ammonium was added to suppress nitrate assimilation, stressing the dissimilatory nature of nitrate reduction. Interestingly, An-4 used intracellular nitrate stores (up to 6-8 μmol NO3 - g-1 protein) for dissimilatory nitrate reduction. Conclusions: Our findings expand the short list of microbial eukaryotes that store nitrate intracellularly and carry out dissimilatory nitrate reduction when oxygen is absent. In the currently spreading oxygen-deficient zones in the ocean, an as yet unexplored diversity of fungi may recycle nitrate to ammonium and nitrite, the substrates of the major nitrogen loss process anaerobic ammonium oxidation, and the potent greenhouse gas nitrous oxide.

Direct observations on chemical micro-environment and microbial composition in biofilms are rare. The combination of microsensor and molecular techniques is highly useful for studies on the microbial ecology of biofilms. We shortly describe some applications of microsensors to study mass transfer phenomena and microbial processes in biofilms. It has recent been recognized that biofilms are not always flat layers of cells, but can consist of complex structures allowing liquid flow. Thus the classical view, that transport in biofilms is diffusional, is challenged. In laboratory grown biofilms the effect of convection on mass transfer was demonstrated. The microsensor technique has improved, so that direct in situ measurements in living biofilms are possible. By direct measurements of liquid flow with microsensors we show that in biofilms grown in bioreactors heterogeneity and convectional transport must also be taken into account. For the description of the microbial population we use molecular techniques, such as in situ hybridisation with 16S rRNA-targeted oligonucleotide probes. In a nitrifying-denitrifying biofilm we found a complex nitrifying community consisting of members of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira. Their occurrence was correlated with nitrification activity as determined by microsensor measurements.

The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 microm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a short-term incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitrite-oxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O 2 , NH 4 + , NO 2 − , and NO 3 − demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter , respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis . 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O2, NH4+, NO2−, and NO3− and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift from an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 μm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Proteobacteria and a thus far unknown nitrite oxidising population.

Here we present preliminary data on the development of a biofilm from a wastewater treatment plant studied with microsensors and molecular techniques. The development during biofilm growth of oxygen, sulfide and pH profiles was measured with microsensors. Anoxic zones developed within one week and further increased during the following weeks. However, sulfide production was delayed and was first detected in a six-week-old biofilm. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments the sequence of the bacterial community was followed showing an increasing complexity of the biofilm community during development. In a mature biofilm the influence of nitrate on sulfide production was studied by measuring oxygen, sulfide, pH, nitrite and nitrate profiles with microsensors. Sulfide production was detected deeper in the biofilm and in lower concentrations, when nitrate was added to the medium. The DGGE pattern of the mature biofilm showed both differences and similarities with the DGGE pattern of the 12-week-old biofilm. In particular the RNA pattern changed when nitrate was added to the medium, indicating a change in activity of certain strains.

We found anoxic zones in aerated activated sludge flocs, and demonstrated denitrification under normal operating conditions. Sulfate reduction was not found. Micro-environments and microbial conversions in flocs from bulking and non-bulking activated sludge were determined with microsensors for H2S, O-2, NO2- and NO3-. Denitrification and sulfate reduction rates were measured with N-15- and S-35-tracer techniques. We showed that under normal reactor conditions (ca. 20% air saturation) anoxic zones develop within flocs allowing denitrification. The denitrification rates amounted to 40% of the rates under anoxic conditions. At 100% air saturation no anoxic zones were found and no denitrification occurred. However, in flocs from bulking sludge (at 20% air saturation) anoxic zones were absent and denitrification did not occur. In bulking sludge only at total anoxia was denitrification found. Confocal microscopy showed that flocs from bulking sludge were much looser than those from non-bulking sludge. The absence of anoxic zones and of denitrification was attributed to the open flee structure, allowing advective oxygen transport. Sulfate reduction was not detected in any of the sludges tested by microsensors or by tracer techniques even under anoxic conditions. This indicates that the sulfur cycle (sulfate reduction and sulfide oxidation) does not play a role in mineralization processes and bulking in activated sludge. Preliminary molecular work tin situ hybridization with the 16S-rRNA probe SRB385) indicated the presence of small amounts of sulfate reducing bacteria in all sludges. Either the probe is not specific or the sulfate reducers present are not active under reactor conditions. (C) 1998 IAWQ. Published by Elsevier Science Ltd.

A highly selective liquid membrane nitrite microsensor based on the hydrophobic ion-carrier aquocyanocobalt(III)-hepta(2-phenylethyl)-cobrynate is described. The sensor has a tip diameter of 10 to 15 (mu)m. The response is log-linear in freshwater down to 1 (mu)M NO(inf2)(sup-) and in seawater to 10 (mu)M NO(inf2)(sup-). A method is described for preparation of relatively large polyvinyl chloride (PVC)-gelled liquid membrane microsensors with a tip diameter of 5 to 15 (mu)m, having a hydrophilic coating on the tip. The coating and increased tip diameter resulted in more sturdy sensors, with a lower detection limit and a more stable signal than uncoated nitrite sensors with a tip diameter of 1 to 3 (mu)m. The coating protects the sensor membrane from detrimental direct contact with biomass and can be used for all PVC-gelled liquid membrane sensors meant for profiling microbial mats, biofilms, and sediments. Thanks to these improvements, liquid membrane sensors can now be used in complex environmental samples and in situ, e.g., in operating bioreactors. Examples of measurements in denitrifying, nitrifying, and nitrifying/denitrifying biofilms from wastewater treatment plants are shown. In all of these biofilms high nitrite concentrations were found in narrow zones of less than 1 mm.

A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. (c) 1997 John Wiley & Sons, Inc.

A dynamic model for growth and substrate consumption of immobilized cells is evaluated by comparing experimental and calculated psendo-steady-state oxygen-concentration profiles in the support material. For this, Nitrobacter agilis cells were immobilized and cultivated in agar slabs. The system was operated as a continuous submerged-bed reactor. At different growth states oxygen-concentration profiles were determined in the slabs with a microsensor. It was shown that simulated and measured profiles agreed very well, both in steady and in pseudo-steady states.

An aerobic biofilm was characterized using confocal scanning laser microscopy (CSLM), O2 micro-electrodes, particle tracking and microinjection of fluorescent dyes. The biofilms were found to consist of microbial clusters of cells and Extra-cellular Polymeric Substance (EPS) separated by interstitial voids. The cell clusters were ca 300 μm and the voids were ca 100 μm wide. The voids were open channels connected with the bulk fluid. Fluorescein micro-injection showed that liquid could flow through the voids, but was always stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, while in cell clusters transport is determined by diffusion only. Particle tracking with CSLM showed that flow velocity inside the biofilm was proportional to the bulk flow velocity. The importance of convective mass transport in biofilms was demonstrated by oxygen distribution measurements. At high flow velocities of the bulk liquid, the mass boundary layer followed the irregular biofilm surface. At lower velocities the mass boundary layer was parallel to the substratum. Mass transfer from voids to cell clusters increased with flow velocity, as result from vonvective mass transport from the bulk to the voids. Convective transport was insignificant at low flow velocities, but at high flow velocities it increased the total mass transport by 200-250%. The local diffusion coefficients in biofilms were measured using microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using CSLM. The diffusion coefficient of small, non-binding molecules in cell clusters is close to that in water. Very large molecules were impeded in their diffusion through the biofilm matrix. It was calculated that the cell cluster matrix had the characteristics of a gel network with pore diameters of 80 nm.

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.

Liquid flow was studied in aerobic biofilms, consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. Fluorescein microinjection was used as a qualitative technique to determine the presence of flow in cell clusters and voids. Flow velocity profiles were determined by tracking fluorescent latex spheres using confocal microscopy. Liquid was flowing through the voids and was stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, whereas in cell clusters diffusion is the dominant factor. The flow velocity in the biofilm depended on the average flow velocity of the bulk liquid. The velocity profiles in biofilms were linear and the velocity was zero at the substratum surface. The velocity gradients within biofilms were 50% of that near walls without biofilm coverage. The influence of the biofilm roughness on the flow velocity profiles was similar to that caused by rigid roughness elements.

Aerobic biofilms were found to have a complex structure consisting of microbial cell clusters (discrete aggregates of densely packed cells) and interstitial voids. The oxygen distribution was strongly correlated with these strutures. The voids facilitated oxygen transport from the bulk liquid through the biofilm, supplying approximately 50% of the total oxygen consumed by the cells. The mass transport rate from the bulk liquid is influenced by the biofilm structure; the observed exchange surface of the biofilm is twice that calculated for a simple planar geometry. The oxygen diffusion occurred in the direction normal to the cluster surfaces, the horizontal and vertical components of the oxygen gradients were of equal importance. Consequently, for calculations of mass transfer rates a three-dimensional model is necessary. These findings imply that to accurately describe biofilm activity, the relation between the arrangement of structural components and mass transfer must be undrstood. (c) 1994 John Wiley & Sons, Inc.

Correction to: The ISME Journal (2010) 4, 417–426; doi:10.1038/ismej.2009.127 Since the publication of this paper, the authors have noticed an error in the list of co-authors and affiliations. The correct list of co-authors and affiliations is shown below. Hang Gao1,2,3, Frank Schreiber1, Gavin Collins1,4, Marlene M Jensen1,5, Olivera Svitlica1, Joel E Kostka1,6, Gaute Lavik1, Dirk de Beer1, Huai-yang Zhou2,7 and Marcel MM Kuypers1

The use of immobilized cells is advocated for a number of bioengineering purposes, e.g. volumetric production rate, retention of expensive biomass, product quality, and convenient downstream processing. Up to now, succesfull application of such biofilms is rather empirical, since detailed knowledge of the internal structure and performance usually is not available. Due to inhomogeneous biomass density, variable kinetics, limited substrate penetration, starvation, high internal product concentrations and inhibitory effects, mathematical modelling of concentration gradients in active bioflims is of limited value. Therefore, direct measurements inside biofilms are needed to verify these models and to improve the mechanistic understanding of biofilm processes. The spatial resolution of classical analytical techniques, such as concentration measurements in extracted pore water from sliced biofilms, amounts to about 1 mm and is insufficient for biofilms with high activity and a concomitant substrate penetration depth of about 100 jim. Microelectrodes, i.e. needle-shaped devices with a sensitive tip, have proven to be most suitable and indispensable tools for these kind of measurements. Interesting studies with oxygen microelectrodes have been performed in e.g. penicillium pellets (Wittier et al., 1986) and trickling filter biofilms (Kuenen et al., 1986), revealing internal convective flows and local oxygen production by algae, respectively.

Abstract 1913 Poster Board I-936 Background: Myeloproliferative neoplasms (MPN) are clonal disorders with an origin of the disease in a hematopoietic stem or progenitor cell. Except for chronic myeloid leukemia (CML), the diagnosis for Philadelphia-chromosome negative (Ph-neg.) MPN is less straightforward. Although many patients with a Ph-neg. MPN can be identified by mutations in JAK2 and/or TET2, the categorization into primary or secondary cythosis or specifically into polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) can be difficult. Since telomeres can be used to estimate the mitotic history of cells, our aims were to evaluate 1) whether the telomere length can be used to distinguish clonal from polyclonal hematopoiesis, 2) whether telomere attrition correlates to the mutational status of JAK2 and 3) whether the extent of telomere shortening in different subsets of leukocytes can point to the originating level of the hematopoietic stem or progenitor cell. Patients and Methods: So far, 32 patients with Ph-neg. MPN diagnosed according to WHO criteria (range: 32 – 85 years; PV: n=19, ET n=5, PMF n=8; JAK2V617F positive n=22, JAK2V617F negative n=16) and 11 patients with secondary erythrocythosis (range 39 – 61 years) were included in this study. 400 healthy individuals (range 0-102 years) served as controls. After informed consent peripheral blood was taken from the patients to measure the telomere length in subsets of leukocytes by automated multicolor flow-FISH. In order to correct for the age-dependent decline in telomere length, telomere length differences to the 50th percentile of the healthy cohort (DeltaTel) were calculated. Telomere length values below the 10th percentile of those from healthy donors were considered as substantially affected by telomere attrition. The mutational status of the JAK2V617F was assessed by allele-specific real time quantitative PCR. Results: The mean telomere length in granulocytes from patients with MPN was considerably shorter compared to healthy controls (mean ± STD DeltaTel: 2.73kb ± 1.20kb), whereas there was no remarkable difference in lymphocytes (0.70kb ± 0.81kb). Furthermore, we found significant DeltaTel between granulocytes from patients with MPN and with secondary erythrocythosis (mean ± STD: 2.73kb ± 1.20kb vs. 1.66kb ± 0.894kb, p Conclusion: In our ongoing study we were able to confirm shorter telomeres in granulocytes of MPN patients compared to telomeres in granulocytes of healthy controls and of patients with secondary erythrocythosis. Based on the telomere length attrition patients with a clonal cythosis can be distinguished from such patients with a secondary cythosis, which could help diagnostically in uncertain cases. No correlation was found between the extent of telomere attrition and the JAK2V617F mutational status. The extremely short telomeres found in most subsets of leukocytes from patients with PMF could point to a very early hematopoietic stem cell as the cell of origin, whereas for ET and PV with only one or a few subsets of leukocytes affected by telomere attrition the cell of origin could be a hematopoietic stem cell at a somewhat later stage. Disclosures: No relevant conflicts of interest to declare.

Abstract 2647 Poster Board II-623 Background/Aims: Reflecting their proliferative history, malignant cells generally have shorter telomeres compared to their normal counterparts. We have reported that leukemic cells from patients with T-prolymphocytic leukaemia (T-PLL) have exceptionally short telomeres (telomere lengths around 1 kb) compared to other malignancies, and high levels of telomerase activity (Roeth et al., Leukemia, 2007). We have also previously presented (Roeth et al., ASCO 2008) cytotoxic effects of the telomerase inhibitory agent imetelstat sodium (GRN163L), which is currently in clinical trials. The objective of this study was to further characterize the relationship between mean telomere length (TL), the single chromosome telomeric size distributions (single TL), telomerase levels and imetelstat-induced telomerase inhibition and cytotoxicity in T-PLL, B-CLL, and normal cells. Methods: Cells were obtained from peripheral blood of normal donors and patients with T-PLL and B-CLL and cryopreserved. TL was measured by flow-FISH (which yields a multicellular average measurement across all chromosomes) and the distribution of individual telomere lengths by single telomere length analysis (STELA, XpYp specific). Telomerase activity (TA) was assessed by TRAP. Effects of imetelstat upon cell survival, apoptosis and TA were studied in short-term cell cultures. Apoptosis was measured by Annexin V-PI double staining. Results: Average (±SD) TL of the T-PLL cells was 1.8 ± 0.73 kb (n=13) which was shorter than for B-CLL cells (ZAP-70+/CD38+ CLL: 2.46 ± 1.08 kb (n=30); ZAP-70neg/CD38neg CLL: 5.06 ± 1.76 kb (n=29), Roeth et al, Br. J. Haem., 2008). T-PLL cells were found to have a narrow range of single chromosome telomere lengths, while both subtypes of B-CLL cells demonstrated a broader distribution of telomere lengths. No attrition of mean telomere length was observed over time in two patients with T-PLL who were followed over time, despite continued clinical progression. In contrast, we have observed that most patients with B-CLL do have decreases in telomere length over the course of their disease. Leukemic T-PLL cells exhibited high levels of telomerase activity, whereas there was little or no detectable telomerase activity in normal, unstimulated T-cells and most B-CLL cells. Imetelstat (GRN163L, Geron Corporation), a potent inhibitor of telomerase, induced significant and dose-dependent inhibition of telomerase activity in T-PLL cells after 2 hours of culture at 1μM and 10μM. Imetelstat significantly reduced cell viability of T-PLL cells in vitro after 7-10 days of culture (viability as % of control (mean ± SD) with 1, 3, 10 μM imetelstat: 69.9 ± 27.3, 51.7 ± 30.2, 37.1 ± 27.6). No significant cytotoxicity of imetelstat was observed in CLL samples, or in unstimulated and stimulated T-cells from 5 normal donors. Leukemic cells from one T-PLL patient early in the course of the disease did not exhibit loss of viability when exposed to imetelstat at levels up to 10 μM; however, when cells from this patient were tested later during the progression of the disease significant cytotoxicity was observed. Conclusions: In summary, leukemic T-PLL cells exhibit extremely short telomere lengths, narrower ranges of single chromosome telomere size distributions, and higher telomerase activity compared to B-CLL cells and normal T cells. These observations may explain why inhibition of telomerase activity in T-PLL cells by imetelstat is associated with rapid and dose-dependent cell death. Disclosures: Baerlocher: Geron Corporation: Research Funding. Bassett:Geron Corporation: Employment, Equity Ownership. Elias:Geron Corporation: Employment, Equity Ownership. Go:Geron Corporation: Employment, Equity Ownership. Roeth:Geron Corporation: Research Funding.

Abstract 3239 Poster Board III-176 Background The Ku autoantigen is a heterodimer composed of a 70kDa subunit (Ku70) and an 80kDa subunit (Ku86), which is critical for repairing DNA double-strand breaks (DSB) by non-homologous end joining and for telomere maintenance. Knockdown of either one or both Ku components results in rapid shortening of telomere ends and genomic instability (Fattah et al., DNA Repair 2008; Faure et al., DNA Repair 2008). We and others have demonstrated significantly shorter telomeres in patients with chronic lymphocytic leukemia (CLL) (Röth A et al, Brit. J. Haematol. 2008). In addition, in over 80% of patients with CLL cytogenetic aberrations are found (Döhner H, NEJM, 2000). Based on these observations we hypothesized that differences in Ku protein expression or function could be involved in the pathophysiology of CLL cells. Our aim was to assess the localization and expression levels of the Ku components in leukemic cells of patients with CLL Patients and Methods So far, we examined the localization and expression of the Ku-proteins in 20 patients with CLL (14 with mutated IgVH genes, 6 with unmutated IgVH genes), and in peripheral blood lymphocytes of 4 healthy individuals. The localization of the Ku proteins was assessed by immunological staining and fluorescence microscopy. Expression levels of the Ku components in whole cells as well as in the cytoplasmic and nuclear fractions were assessed by Western blotting. DNA sequencing was done by cycle sequencing and capillary electrophoresis. Telomere length measurements were performed by automated multicolor Flow-FISH. Results Lymphocytes of healthy individuals with a normal telomere length showed an expression of Ku70 and Ku86 in the nucleus without any cytoplasmic expression. Interestingly, in leukemic cells from CLL patient samples, Ku86 was not detectable in the nucleus, but was abundantly found in the cytoplasm, while Ku70 was found in approximately equal amounts in the nucleus and cytoplasm. The expression levels of both Ku proteins were comparable in lymphocytes from CLL patients and from healthy controls. Sequence analysis of the Ku70 and Ku86 DNA revealed no mutations. All leukemic cells of CLL patients had short telomeres below the 50th percentile of healthy individuals with significantly shorter telomeres in patient samples with unmutated IgVH (mean: 2.8kb ± 0.03kb) than with mutated IgVH (mean: 6.15kb ± 2.92kb). In contrast, no difference in the expression pattern of Ku70 and Ku86 was found between these two prognostic subgroups of CLL patients. Conclusion For the first time we can demonstrate clear differences in the expression and localization of the Ku proteins in leukemic cells of patients with CLL with short telomeres compared to lymphocytes of healthy individuals with telomeres in the normal range. The high cytoplasmic expression of both Ku proteins in the leukemic cells of CLL patients and the lack of nuclear Ku86 protein suggested the presence of a genetic alteration. Although we could not find any mutations in the DNA sequence of the Ku proteins, the delocalization could point to changes in the Ku proteins (i.e. by post-translational modifications) and/or in the transport system of the Ku proteins between the nucleus and the cytoplasm. Deficient nuclear expression of Ku86 and/or Ku70 in CLL cells might lead to shortening of telomeres and genomic instability as has been seen in heterozygous Ku86 deficient human cell lines. Since there was no difference in the expression and localization of the Ku proteins in CLL cells with mutated and unmutated IgVH it is unlikely that the absolute telomere length, which differs highly between the two subgroups of CLL patients, is directly related to the level of the nuclear Ku protein expression. Further molecular and functional studies of the Ku proteins are ongoing. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: In chronic lymphocytic leukemia (CLL) short telomeres were shown to be associated with mutational status, progression free survival (PFS) and overall survival (Damle et al., 2004). Chromosomal instability increases with shortening of telomeres. Recently, a relationship between telomere length and number of chromosomal aberrations has been shown if telomere length was investigated by quantitative real-time polymerase chain reaction (Tel-PCR) (Roos et al., 2008). The aim of the present study was to correlate average telomere length of individual cells measured by multicolor flow-FISH to established prognostic factors and genomic aberrations. PATIENTS and METHODS: Blood samples from 64 patients with CLL were analyzed. Flow cytometry was performed for quantification of ZAP-70 and CD 38 expression with a cut off at 20%. Immunoglobulin variable heavy chain (IGVH) genes were sequenced. An IGVH gene sequence with less than 98% homology with the corresponding germ-line sequence was considered to be mutated. Chromosomal alterations were investigated by fluorescence in situ hybridization (FISH) with the following gene probes: LSI 13q14, LSI 13q34, CEP 12, LSI 17p13, LSI 11q22–23. Copy number changes were also detected in 18 samples by SNP-chip analysis. The average length of telomere repeats at chromosome ends was measured by multicolor flow-FISH. Values of telomere length from CLL cells were correlated to values of telomere lengths of B lymphocytes from healthy age matched individuals (delta telomere length=Δtel). RESULTS: The average telomere length of the clonal B-cells was short. Patient samples from advanced Binet stages (B/C) had significantly shorter telomeres (Δtel −4.8 ± 1.0 kb) than patients samples from Binet A (Δtel −3.4 ± 1.2 kb, p=0.03). The average telomere length was significantly shorter for ZAP-70+ (Δtel −5.0 ± 0.5 kb) and CD38+ (Δtel −4.9 ± 0.7 kb) patient samples than for ZAP-70− (Δtel −2.4 ± 0.8 kb) and CD38− (Δtel −3.0 ± 1.0 kb) patient samples, respectively (p DISCUSSION: We are able to confirm significant shorter telomeres in CLL samples with unfavourable prognostic factors like advanced Binet stage, positivity for ZAP-70 or CD38 and unmutated IGVH genes compared to their favourable counterparts. CLL samples with the chromosomal aberrations del 17p and del 11q are associated with bad clinical outcome. CLL with these aberrations of the present study demonstrated significantly shorter telomeres compared to cases without these abnormalities. Additional studies relating impact of telomere length on genomic instability detected by SNP-chip are ongoing.

BACKGROUND: In contrast to human granulocytes and T-cells with a quite homogenous decrease in telomere lengths over age, telomere lengths of peripheral blood B-cell populations are highly heterogeneous with average telomere lengths of B-cells remaining relatively constant from middle age onward (Baerlocher et al, J. Leuk. Biol. 2003). So far, telomere elongation has been described in highly proliferating germinal center B-cells during the development from naive B-cells to memory B-cells (Norrback et al, Eur J Haematol 2001). During this same time period the process of class-switch recombination (CSR) occurs. Our aims were to analyze the telomere lengths in different B-cell subsets in order to elucidate telomere length dynamics in relationship to CSR. PATIENTS and METHODS: Buffy coats from 14 healthy donors were enriched for CD19+ B cells by magnetic cell separation. Naive B cells (IgM+/IgD+/CD27−), IgM-only B cells (IgM+/IgD−/low/CD27+), double-positive B cells (IgM+/IgD+/CD27+) and class-switched memory B cells (IgM−/IgD−/CD27+) were further separated by cell sorting. Telomere length of sorted B-cell subpopulations was measured by automated multicolour flow-FISH. RESULTS: Naive B-cells presented the shortest telomere length values (average telomere length, n=14: 7.2 kb ± 0.7 kb) compared to the other B-cell subsets. In comparison to the naive B-cell subset, the IgM only B-cell subset had 13% longer telomeres (average telomere length, n=14: 8.1 kb ± 1.3 kb), the double-positive B-cell subset had 10% longer telomeres (average telomere length, n=14: 7.9 kb ± 0.8 kb) and the memory B-cell subset had 22% longer telomeres (average telomere length, n=14: 8.7 kb ± 1.1 kb) (p < 0.0001). CONCLUSIONS: We are able to confirm longer telomeres in memory B cells than in naive B cells. For the first time, however, we can demonstrate that longer telomeres are also found in non-class switched B-cells. Based on these results telomere elongation does not coincide with the process of CSR. Additional studies are needed to assess whether telomere elongation can only take place in the germinal center or whether certain B-cell subsets are able to elongate their telomeres independent from the germinal center.

BACKGROUND: In contrast to other B-cell neoplasias, chronic lymphocytic leukemia (CLL) is not only characterized by a clonal expansion of specific B-cells, but also by an increase in non-leukemic T-cells, most likely involved in sustaining the growth of the leukemic B-cell clone. Based on ZAP-70, CD38 and the IgVH mutation status, two prognostic groups of CLL patients can be identified. Our aim was to characterize the replicative histories of the B- and T-cells in the two groups of CLL patients compared to healthy individuals. PATIENTS and METHODS: Blood samples from 73 patients with CLL (ZAP-70−/CD38−: n = 29, ZAP-70+/CD38+: n = 30, ZAP-70/CD38 discordant: n = 14) were analyzed. The quantity and characteristics of the lymphocyte subsets was assessed by a cell counter and by immunophenotypic analysis. The replicative histories of naive and memory T-cells as well as B-cells was determined by measurements of telomere length in peripheral blood leukocytes of CLL patients and healthy individuals by automated multicolor flow-FISH. RESULTS: As expected, the average telomere length of the clonal B-cells was short. The telomere length was, however, significantly shorter for the ZAP-70+/CD38+ patient samples (2.46 ± 1.08 kb) than for the ZAP-70−/CD38− patient samples (5.06 ± 1.76 kb, p < 6.7 x 10−9). Interestingly, also the naive and memory T-cells from ZAP-70+/CD38+ CLL patients exhibited significantly shorter average telomere lengths (mean ± std: 4.85 ± 1.58 kb; 4.39 ± 1.09 kb) than T-cells from ZAP-70−/CD38− CLL patients (6.64 ± 1.72 kb, p < 2.2 x 10−4; 6.22 ± 1.5 kb, p < 7.4 x 10−6). These results are in line with the observed higher absolute T-cell numbers in the ZAP-70+/CD38+ CLL patients compared to ZAP-70−/CD38− CLL patients. Moreover, the average telomere loss in relation to time from primary diagnosis to sample date was higher for naive T-cells than memory T-cells in ZAP-70+/CD38+ patients (7.8 vs. 5.8 bp/month). When we compared the telomere length to age-related percentiles calculated from over 400 healthy individuals aged 0–102 years practically all telomere length values of the naive and memory T-cells from the ZAP-70+/CD38+ CLL patients fell below the 50th percentile, whereas the values of naive and memory T-cells from the ZAP-70−/CD38− CLL patients were within the normal distribution. CONCLUSIONS: We can confirm significantly shorter telomere length values for the B-cells of the ZAP-70+/CD38+ CLL patients. In addition, we can also demonstrate significantly shorter telomeres in T-cells of ZAP-70+/CD38+ CLL patients, which are below the 50th percentile compared to controls, and a higher telomere loss over time for naive T-cells of ZAP-70+/CD38+ CLL patients. As telomere length shortens approximately 50 to 100 bp per cell division the observed decrease in telomere length of the T-cells in ZAP-70+/CD38+ CLL patients equals to approximately 18 to 36 population doublings. This is by far more than expected by the slightly higher T-cell numbers in the peripheral blood. Our observations imply an extensive expansion of the T-cell compartment in ZAP-70+/CD38+ CLL patients and suggest an important role of T-cells in this subgroup of CLL patients.

AT the surface of sulphide-rich marine and freshwater sediments, the gliding filamentous sulphur bacteria Beggiatoa spp. often grow abundantly to form conspicuous white mats1–6. Beggiatoa may be responsible for the whole benthic consumption of oxygen, converting sulphide to either intracellular elemental sulphur, or to extracellular sulphate7–9. Various strains of Beggiatoa have shown a capacity for nitrogen fixation and assimilation of ammonium and nitrate10. We now report that denitrification activity may also be attributed to Beggiatoa spp., which indicates that Beggiatoa mats could have a key role in the benthic cycling of oxygen, sulphur and nitrogen. Microprofiles of nitrate through a Beggiatoa mat on a lake sediment showed that all nitrate taken up from the water phase was consumed within the upper 150 μ m of the mat. Complete denitrification was demonstrated by the conversion of 15NO−3 to 15N2 in semi-purified tufts of Beggiatoa, and the coupling of denitrification and sulphide oxidation was indicated by micro-profiles of oxygen and sulphide in pure cultures.